Molecular mechanisms of coronary artery disease risk at the PDGFD locus

Genome wide association studies for coronary artery disease (CAD) have identified a risk locus at 11q22.3. Here, we verify with mechanistic studies that rs2019090 and PDGFD represent the functional variant and gene at this locus. Further, FOXC1/C2 transcription factor binding at rs2019090 is shown to promote PDGFD transcription through the CAD promoting allele. With single cell transcriptomic and histology studies with Pdgfd knockdown in an SMC lineage tracing male atherosclerosis mouse model we find that Pdgfd promotes expansion, migration, and transition of SMC lineage cells to the chondromyocyte phenotype. Pdgfd also increases adventitial fibroblast and pericyte expression of chemokines and leukocyte adhesion molecules, which is linked to plaque macrophage recruitment. Despite these changes there is no effect of Pdgfd deletion on overall plaque burden. These findings suggest that PDGFD mediates CAD risk by promoting deleterious phenotypic changes in SMC, along with an inflammatory response that is primarily focused in the adventitia.


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Sample size was determined based on extensive experience with similar experiments in our laboratory (PMID: 36246779, PMID: 34990206, PMID: 32441123). Exact sample size for each experiment is indicated in the figure legends for each panel. These sample sizes were sufficient because they provided statistically significant results that were cross-validated with other modalities, for instance single cell RNA sequencing data and histology data for cell number were used for cross-validation.
The number of genes, number of unique molecular identifiers and the percentage of mitochondrial genes were examined to identify outliers. As an unusually high number of genes can result from a 'doublet' event, in which two different cell types are captured together with the same barcoded bead, cells with >6000 genes were discarded. Cells containing >7.5% mitochondrial genes were presumed to be of poor quality and were also discarded. There were no other exclusions.
All attempts of replication were successful. All experiments were performed at least three times, with consistent results. Experiments were conducted with at least three technical replicates, exact number of biological replicates can be found in the figure legends.
For single cell RNAseq of knockout animals, there was no randomization possible. All animals used for the antibody blocking were selected and grouped at random.
Workers were blinded to sample genotype identity for the Pdgfd knockout lesion analysis, and for antibody blockade lesion analyses.
Lesion histology cell areas were determined with Cnn1 rabbit monoclonal primary antibody at 1:400 dilution (TA327614; Origene), and CD68 rabbit polyclonal antibody at 1:300 dilution (ab125212; Abcam). Lots were not recorded. Blocking antibody Pdgfd 25E17, PDGFD-ab was employed in in vitro blocking and in vivo blocking experiments. An IgG1 isotype control antibody was custom developed to serve as a negative control for the Pdgfd blocking antibody.
Testing was performed with slides containing cells of known origin and known CD68 or Cnn1 staining. Pdgfd antibody 25E17 experimental validation is presented in Supplemental Tick this box to confirm that the raw and calibrated dates are available in the paper or in Supplementary Information.

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Primary HCASMC were purchased from Cell Applications, Inc (San Diego, CA) and Lonza BioScience and were cultured in complete smooth muscle basal media (Lonza, #CC-3182) according to the manufacturer's instructions. All experiments were performed with HCASMC between passages 5-8. Rat aortic smooth muscle cells (A7r5) and human embryonic kidney 293 cells (HEK293) were purchased from ATCC and cultured in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Fisher Scientific, #MT10013CV) with 10% FBS at 37 ºC and 5% CO2. A7r5 at passage 6-18 were used for experiments. IMR90 fetal lung fibroblasts at passage 7 were cultured in FGM-2 lung fibroblast basal media (Lonza, #CC-3131) according to the manufacturer's instructions.
Smooth muscle cells are not cell lines, but primary cultured cells sustained in culture, with no available genetic information, so authentication regarding their authenticity is not a concerns. These cells were authenticated to be human smooth muscle cells by gene expression and morphology and phenotypic changes with stimulations. Other cell lines were validated by RT-PCR for a panel of expressed genes.
Testing was regularly performed in all cell populations studied.
No commonly misidentified lines were used in these studies.
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We used a Pdgfd constitutive null line (Pdgfdtm1Ueks) on the C57Bl/6 background and was transferred to us by Ulf Eriksson at the Karolinska. The Pdgfd gene, 100 basepairs from the ATG start codon to the 3'-end of exon 1, was replaced by a lacZ reporter cassette. To identify and track cells that originate from the media we used the validated SMC-specific mouse Cre allele (Myh11CreERT2) combined with the floxed tandem dimer tomato (tdTomato) fluorescent reporter protein gene knocked into the ROSA26 locus (B6.Cg-Gt(ROSA)26Sortm14(CAGtdTomato)Hze/J), in the ApoE null background.
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